Expression level is one of the most important considerations in the utilization of cloned gene products. Elevated levels of protein expression have important ramifications both in terms of protein yield per fermentation volume and in degree of purification difficulty. Most efforts at increasing expression of cloned gene products have, to date, focused on the use of strong promoters in conjunction with an efficient ribosome binding site. A variety of promoters have been used to increase expression, the most commonly used being the P.sub.L promoter from phage lambda and the E. coli lacUV5 and trp promoters.
The lambda P.sub.L promoter has been successfully used in conjunction with a CI857 temperature-sensitive lambda repressor. This allows for low level expression of the cloned product during E. coli growth at 30.degree. C. Once substantial cell density is established, the cloned gene can be derepressed by growth at 42.degree. C. This method has been used in the expression of gene products lethal to the host cells. Several investigators have reported expression levels of 4% (Waldman, A. S., Haensslein, E., and Milman, G. [1983] J. Bio. Chem. 258: 11571-11575); 7% (Yoakum, G. H., Yeung, A. T., Mattes, W. B., and Grossman, L. [1982] PNAS 79: 1766-1770; Derom, C., Gheysen, D., and Fiers, W., [1982] Gene, 17: 45-54); and 13% (Oehrnichen, R., Klock, G., Altschmid, L., and Hillen, W. [1984] EMBO J. 3: 539-543) using the P.sub.L promoter under thermolabile repressor control.
Recently, there has been increased use of a chimeric promoter consisting of sequences from the E. coli lacUV5 and trp promoters. This hybrid promoter is known as the tac promoter; it contains the -10 region from the lac promoter and the -35 region of trp. This hybrid promoter is repressed by the E. coli lac I.sup.q gene product and induced by 5 mM isopropyl-.beta.-D-thiogalactopyranocide (IPTG). This system has been used by several investigators with varying results. Expression of various proteins have reached the 7% level (Bagdasarian, M. M., Amann, E., Lurz, R., Ruckert, B., and Bogdasarian, M. [1983] Gene 26: 273-282); the 10% level (Bikel, I., Roberts, T. M., Bladon, M. T., Green, R., Amann, E. and Livingston, D. M. [1983] PNAS 80: 906-910) and the 30% level (Amann, E., Brosius, J., and Ptashne, M. [1983] Gene 25: 167-178).
Protein expression levels are dependent on the genetic background of the host cell. The utilization of host cells containing specific mutations has been shown to increase the level of a cloned protein. Two genes have received wide attention in this regard, the lon and pnp mutations.
The lon mutation has been mapped to the capR region of the E. coli genome and has been shown to code for an ATP-dependent protease (Bukhari, A. I. and Zipser, D., [1973] J. Bact. 116: 1469-1471; Shineberg, B. and Zipser, D., [1973] J. Bac. 116: 1469-1471). This ATP-dependent protease is one of the eight proteases found in E. coli (Chung, C. H. and Goldberg, A. L. [1981] PNAS 78: 4931-4935; Sreedhara Swamy, K. H. and Goldberg, A. L. [1981] Nature 292: 652-654). It has been demonstrated to be the major protease involved in the degradation of proteins produced from missense and nonsense mutations (Mount, D. W. [1980] Ann Rev. Genet. 14: 297-319). The pnp mutation has been mapped to the polyribonucleotide phosphorylase gene. Polyribonucleotide phosphorylase has been shown to be involved in the phosphorolysis of ribonucleic acid and therefore implicated in mRNA breakdown. Subsequent studies have shown a 20- to 100-fold increase in specific activity of cloned fungal catabolite dehydrogenase when cloned into pnp mutant strains (Hautala, J. A., Bassett, C. L., Giles, N. H. and Kushner S. R. [1979] Proc. Natl. Acad. Sci. USA 76: 5774-5778). These studies also demonstrated a 4- to 7-fold increase in plasmid copy number in these mutant strains. Thus the increase in enzyme-specific activity could be due to increased mRNA synthesis, increased mRNA lifetime, or a combination of both phenomena.
The rop (repressor of primer) gene has been known for some time to control plasmid copy numnber. In 1980, it was demonstrated that deletion of a non-essential region of E. coli colEl derived plasmids increases plasmid copy number. Deletion of this region increased plasmid DNA from 4% of chromosomal DNA to 20%. This deletion was trans recessive as coinfection of the host with a wild type plasmid reduced the copy number of the mutant plasmid. (Twigg, A. J. and Sherratt, D. [1980] Nature 283: 216-218)
Recent prior art reports for E. coli expression systems, wherein proteins foreign to the E. coli host are produced, disclose expression levels of about 25 to 30% of total cellular protein. Simons et al. reported that human interferon gamma was expressed at levels up to 25% of total cellular protein. These workers utilized the P.sub.L promoter of phage lambda followed by the translational initiator region derived from either phage MS2 replicase or the E. coli tryptophan attenuator region (Simons, G., Remaut, E., Allet, B., Devos, R. and Fiers, W. [1984] Gene 28: 55-64.) Amman at al. have expressed the lambda repressor as 30% of total cellular protein using the tac promoter system (Amman, E., Brosius, J. and Ptashne, M. [1983] Gene 25: 167-178). As stated above this promoter contains the -10 region of the lacUV 5 promoter and the -35 region of the trp promoter (DeBoer, H. A., Comstock, L. J., Yansura, D. G. and Heynecker, H. L. in Promoters, Structure and Function, Praeger, New York [1982] 462-481 (R. L. Rodriquez and M. J. Chamberlin eds.)